The technique of nucleic acid hybridization has been successfully employed for the study of DNA structure nucleic acid purification, gene localization, and detection and diagnosis of diseases and mutations.
Hybridization assays are based on the structural properties of DNA molecules. The DNA of most organisms is comprised of two strands of polynucleotides which are associated by means of noncovalent interactions (e.g. hydrogen bonding, stacking forces, etc.) into the familiar double helical structure. It was demonstrated by Britten, et al. (Sci. American 222(4): 24-31 (1968)) that under certain conditions it was possible to cause the two strands to separate from one another. This process of strand separation has been variously referred to as unwinding, denaturing or melting of the double-stranded duplex. It was further discovered that under a second set of conditions the strands would reassociate to reform the duplex DNA structure, this process being referred to as reassociation or renaturation. By measuring the kinetics of reassociation, estimates were able to be made of the relative amounts of unique sequence DNA to repetitive or reiterated DNA sequences.
Further studies demonstrated it was possible to denature the DNAs from two different sources (e.g. two different species of bacteria, two different types of animals or plants) then mix the two populations of single stranded nucleic acids and under renaturation conditions estimate the percentage of double stranded hybrids that were formed; such being an indication of sequence homology between the two sources. The double-stranded molecules formed by the reassociation of one strand from a first source and another strand from a second source are known as a hybrid DNA molecules and the process of forming such molecules is known as DNA hybridization. In a related embodiment a small nucleotide segment comprising a fragment of a single gene up to a size which would include several genes may be used to hybridize to DNA sample for the purposes of identifying if a complementary segment exists in the sample as well as its localization within the sample. The segment is often of predetermined sequence or function and is generally referred to as a nucleic acid hybridization probe. These probes have become extremely important as reagents for the detection of specific nucleic acid sequences. Commonly the probes are labelled with radioactive isotopes to facilitate their analytical detection. The isotopes normally employed include .sup.32 P, .sup.125 I or .sup.3 H; however, considerations regarding stability, safety, ease of detection and disposal of waste have fostered the development of non-isotopically labelled probe molecules.
One approach has been to detect nucleic acids by immunological means, either by developing antibodies which will discriminate between single and double stranded DNAs or by labelling the nucleic acid with an immunoreactive component such as a hapten. Landegert, et al. (Exp. Cell Res. 153: 61-72 (1984)) and Tchen et al. (Proc. Nat'l Acad. Sci. USA 81: 3466-3470 (1984)) have employed N-acetoxy-N-2-acetylaminofluorene to develop immunogenic probes the detection of which is by classical direct or indirect enzyme-linked immunosorbent assays (ELISA). Because of the carcinogenic nature and attendant disposal problems associated with N-acetoxy-N-2-acetylaminofluorene, alternative methods are desired.
A hapten which has gained widespread use for labelling nucleic acid molecules is the vitamin, biotin. Of particular advantage is the high affinity (K.sub.d =10.sup.-15 M) displayed for biotin by the glycoprotein avidin (Green, N. M., Adv. Protein Chem. 29: 85-133 (1975)). Subsequently it was found that avidin could be reacted with enzymes, fluorescent groups or electron dense molecules to form analytically detectable avidin-conjugates.
Ward, et al. (Proc. Nat'l Acad. Sci. USA 79: 4381-4385 (1982) and Proc. Nat'l Acad. Sci. USA 80: 4045-4049 (1983)) have developed a method for the enzymatic incorporation of biotin-labelled analogs of dUTP and UTP into nucleic acids. Although these methods have been quite useful, different types of nucleic acids require modifications of the protocol, and the method requires expensive substrates and enzyme which made large scale preparation economically disadvantageous. It was desirable, therefore, to develop chemical methods for labelling nucleic acid with biotin. Several attempts to develop chemical labelling methods have been reported.
Manning et al. (Chromosoma 53: 107-117 (1975)) have disclosed the chemical cross-linking of a biotin labelled cytochrome c conjugate to RNA with formaldehyde. M. Renz and C. Kurz substituted enzymes such as peroxidase or alkaline phosphatase for cytochrome c in a similar cross-linking proce-dure (Nucleic Acid Res. 12(8): 3435-3444 (1984)).
These methods have been associated with problems of instability of the conjugates under hybridization conditions or the steric hindrance of hybridization itself.
Finally, Forster, et al. (Nucleic Acid Res. 13(3): 745-761 (1985)) have disclosed the synthesis of a photo-activatable biotin analog of the formula: ##STR2## which may be used to label M13 DNA probes. However, this compound reacts with both single and double stranded DNA and as pointed out by the authors, this dual reactivity limits the extent of probe modification possible without interfering with the hybridization of target sequences by single stranded regions of the probe.